Stimulus activation of olfactory receptor neurons is initiated by reversible interactions of odorants with receptor proteins in the membranes of cilia projecting from the dendritic ends of the cells. Stimulus- receptor interaction triggers a sequence of events leading to membrane depolarization and discharge of action potentials. It is now generally accepted that G-protein-linked second messengers are involved in coupling receptor activation with changes in membrane conductance. Particularly convincing evidence has been obtained for coupling of at least some olfactory receptors to G-protein-linked formation of cAMP, which in turn directly activates cation channels in ciliary membranes. Recent biochemical and electrophysiological evidence indicates that, in catfish and rat olfactory receptor cells, some odorants trigger a G-protein-coupled increase in inositol-1,4,5-trisphosphate (IP3), and that IP3 directly activates relatively nonselective calcium channels in cilia membranes. The major goal of the proposed research is to clarify the relative roles of cAMP and IP3 in olfactory signal transduction in two species, the catfish and the rat. Biochemical, electrophysiological and biophysical methods will be used to define the molecular mechanisms that link stimulus-receptor interaction with subsequent ionic events in olfactory neurons. Radioligand binding and second messenger formation will be measured in the millisecond time range to correlate the kinetics of second messenger formation with levels of receptor occupancy. Stimulus-induced changes in intracellular calcium levels will be monitored in real time using fura-2 and video microscopy. These changes will be correlated with the type of odorant and the second messenger pathway activated. Second messenger-regulated changes in membrane conductance will be examined in intact cells using whole-cell recording techniques and single-channel properties will be studied in excised patches and planar bilayers into which ciliary membrane fragments have been incorporated. Application of these techniques to the catfish model system, in which stimulus-receptor interaction is well characterized, will provide a comprehensive description of the mechanisms of olfactory signal transduction in this species, as well as the basis for studies in mammalian receptor neurons.